Method of decomposing protein with sulfonic acid compound

ABSTRACT

A method of assaying a glycated protein in a sample with the use of redox reaction, in which highly reliable measurement can be obtained. A sample containing a glycated protein is treated with protease in the presence of a sulfonic acid compound, so that the glycated protein is degraded. The glycated portion of the resultant glycated protein degradation product is reacted with fructosyl amino acid oxidase, and this redox reaction is measured, thereby determining the amount of glycated protein. Sodium lauryl sulfate can be used as the sulfonic acid compound.

TECHNICAL FIELD

The present invention relates to a method of degrading proteins, and amethod of treating glycated proteins in a sample with a protease anddetermining the amount of the glycated proteins using a redox reaction.

BACKGROUND ART

Conventionally, in order to detect a target protein (including apeptide) in a sample or deactivate a function of the protein thataffects measurements, a method of degrading the protein with a proteasehas been applied in various measuring methods.

For example, an attempt is now being made to measure, by an enzymaticmethod, glycated proteins in blood cells, which serve as a significantindicator in the diagnosis, therapy and the like of diabetes,especially, glycated hemoglobin in erythrocytes, which reflects thepatient's past history of blood glucose levels. In this case, the methodof degrading the glycated proteins with the protease also is used. Inthe enzymatic method, a fructosyl amino acid oxidase (hereinafter,referred to as “FAOD”) is allowed to act on a glycated portion of theglycated protein in a hemolyzed sample, thus generating hydrogenperoxide. The amount of this hydrogen peroxide corresponds to the amountof the glycated protein. Then, a peroxidase (hereinafter, referred to as“POD”) and a substrate that develops color by oxidation are added to thesample that has been treated with FAOD, so that a redox reaction occursbetween the hydrogen peroxide and the substrate with the POD as acatalyst. At this time, since the substrate develops color when it isoxidized, the amount of the hydrogen peroxide can be determined bymeasuring the color developed. As a result, the amount of the glycatedprotein in the sample can be determined.

However, the FAOD to be allowed to act on the glycated portion acts on aglycated amino acid and a shorter glycated peptide fragment more easilythan on a glycated protein and a glycated peptide. Accordingly, bydegrading the glycated protein and the glycated peptide in advance witha protease so that the FAOD can act on the glycated portion more easily,the accuracy of measurement is improved.

However, since the protease has a substrate specificity and showsdifferent degrading activities depending on the substrate to be treated,there is a problem that, depending on the kind of a target protein, thedegradation may take a long time and the measurement cannot be carriedout quickly. Further, when the target protein is glycated as in the caseof the above-mentioned glycated protein, it sometimes is difficult todegrade due to its steric hindrance and the like. For this reason, whenmeasuring the glycated protein in the above-mentioned manner, forexample, the measurement process as a whole also takes a long time owingto the protease treatment carried out in advance. Therefore, from theviewpoint of applicability in the field of clinical tests etc., therehas been a demand for a method by which the glycated protein can bemeasured more quickly.

DISCLOSURE OF INVENTION

Accordingly, it is an object of the present invention to provide amethod of producing a protein degradation product by which a protein isdegraded quickly and efficiently, and a method of degrading a glycatedprotein in a sample and determining the amount of the glycated protein.

In order to achieve the above-mentioned object, a method of producing aprotein degradation product according to the present invention includestreating a protein with a protease in the presence of a sulfonic acidcompound. In the present invention, the “protein” also includes apeptide, and the “protein degradation product” includes a degradationproduct of the above-mentioned peptide.

The protease treatment in the presence of the sulfonic acid compound inthis manner makes it possible to degrade a protein in a sample quickly.

Next, a method of measuring a glycated protein according to the presentinvention, in which an amount of the glycated protein is determined bytreating a sample containing the glycated protein with a protease so asto degrade the glycated protein, allowing a glycated portion of aglycated protein degradation product obtained by the degradation and aFAOD to react with each other, and measuring this redox reaction,includes carrying out the protease treatment in the presence of asulfonic acid compound. Incidentally, the “glycated protein” in thepresent invention also includes a glycated peptide.

In a conventional method carried out in the absence of a sulfonic acidcompound, a protease treatment as long as, for example, about 6 to 40hours has been necessary for degrading the glycated protein sufficientlywith the protease so as to allow FAOD to act on the glycated portioneasily. Accordingly, the above-mentioned enzymatic method requires along time to measure the glycated protein and thus is not very useful.In contrast, according to the measuring method of the present invention,since it is possible to degrade a glycated protein or the like within ashort period, a glycated protein or the like can be measured quickly.For example, under the same conditions as in the conventional methodexcept that a sulfonic acid compound is added, the measuring method ofthe present invention can shorten the time required for the measurementto about 1/10 to 1/2000 of that in the case where no sulfonic acidcompound is present. Consequently, the measuring method according to thepresent invention achieves a still quicker and more accuratemeasurement, which is useful for various tests in clinical medicine asdescribed above.

BEST MODE FOR CARRYING OUT THE INVENTION

In the method of producing a protein degradation product and the methodof measuring a glycated protein according to the present invention, thesulfonic acid compound can be a compound represented by, for example, ageneral formula: R—SO₃X.

In the above formula, X is, for example, Na, K, Li, H or the like, and Rpreferably is a hydrophobic group, for example, CH₃(CH₂)_(n)—,CH₃(CH₂)_(n)—C₆H₄—, C₆H₅—, C₆H₅—N═N—C₆H₄—, C₆H₅—CH═CH—C₆H₄— or the like.For example, n in the above R ranges from 1 to 20. In the above R, “H”may be substituted by an acyl group, a nitro group, a nitroso group, aphenyl group, an alkyl group, an alkyl ether group or the like.

Specific examples of the sulfonic acid compound include, for example,sodium lauryl sulfate (hereinafter, referred to as “SLS”),dodecylbenzenesulfonic acid sodium salt (hereinafter, referred to as“SDBS”), lithium lauryl sulfate (hereinafter, referred to as “LiLS”),4-aminoazobenzene-4′-sulfonic acid sodium salt (hereinafter, referred toas “ABSA”), 4-amino-4′-nitrostilbene-2,2′-disulfonic acid disodium salt(hereinafter, referred to as “ANDS”),4,4′-diazidostilbene-2,2′-disulfonic acid disodium salt (hereinafter,referred to as “DADS”), N-cyclohexyl-2-aminoethane sulfonic acid,N-cyclohexyl-3-aminopropane sulfonic acid,N-cyclohexyl-2-hydroxy-3-aminopropane sulfonic acid,piperazine-1,4-bis(2-ethane sulfonic acid), bathophenanthroline sulfonicacid and the like, and the sulfonic acid compound more preferably isSLS, SDBS or LiLS.

Since these sulfonic acid compounds generally have a high solubility,they can be treated easily even when the concentration of glycatedproteins in the sample is high. Also, in view of their inexpensiveness,the sulfonic acid compounds are very useful.

Further, in the producing method and the measuring method according tothe present invention, it is preferable to carry out a proteasetreatment in the presence of both the sulfonic acid compound and a nitrocompound because the degradation of the glycated protein can beaccelerated further.

The above-noted nitro compound is not particularly limited but can be,for example, a nitrobenzene compound or a dinitrobenzene compound. Abenzene ring of these compounds preferably has not only the nitro groupbut also a substituent such as —NH₂, —OH, —COOH, —SO₃ or —(CH₂)_(n)CH₃(n=2 to 9). The substituent also can be, for example, a halogen group,an ether group or a phenyl group.

Specific examples of the nitro compound include, for example,2,4-dinitrophenol (2,4-DNP), 2,5-dinitrophenyl, 2,6-dinitrophenyl,4,6-dinitro-2-methyl phenol, 2-amino-4-nitrophenol,2-amino-5-nitrophenol, 2-amino-4-nitrophenol, p-nitrophenol (p-NP),2,4-dinitroaniline (2,4-DNA), p-nitroaniline (p-NA), sodium nitrite(NaNO₂), potassium nitrite (KNO2),4-amino-4′-nitrostilbene-2,2′-disulfonic Acid Disodium Salt(hereinafter, referred to as “ANPS”), nitrobenzene and the like. Whenboth of the sulfonic acid compound and the nitro compound are present asdescribed above, the combination thereof is not particularly limited.

In the producing method and the measuring method according to thepresent invention, the protease is not particularly limited but can be,for example, serine proteases, thiol proteases, metalloproteinases orthe like. More specifically, it is preferable to use trypsin, proteinaseK, chymotrypsin, papain, bromelain, subtilisin, elastase, aminopeptidaseand the like. In the case where the glycated protein to be degraded isglycated hemoglobin, it is more preferable to use as the protease aprotease degrading the glycated hemoglobin selectively, e.g., bromelain,papain, trypsin derived from porcine pancreas, metalloproteinase andprotease derived from Bacillus subtilis. Examples of the proteasederived from Bacillus subtilis include Protease N (trade name,manufactured by Fluka Chemie AG, for example), Protease N “AMANO” (tradename, manufactured by Amano Enzyme Inc.) and the like. Examples of themetalloproteinase include the metalloproteinase (EC 3. 4. 24. 4) derivedfrom the genus Bacillus and the like. Among these, metalloproteinase,bromelain and papain are more preferable, and metalloproteinase is mostpreferable. By using the protease that allows a selective degradation asabove, a degradation product of a specific glycated protein can beprepared selectively.

As described above, the method of producing a protein degradationproduct according to the present invention is characterized by treatinga protein with a protease in the presence of a sulfonic acid compound.

The amount of the sulfonic acid compound to be added is not particularlylimited but can be determined suitably according to, for example, thekind and added amount of the protease, the kind of sample, the amount ofproteins contained in the sample or the like.

More specifically, the sulfonic acid compound is added preferably in therange of 0.01 to 1000 μmol, more preferably in the range of 0.03 to 200μmol and particularly preferably in the range of 0.05 to 40 μmol withrespect to 1 μL of the sample.

In the case of adding both the sulfonic acid compound and the nitrocompound, the sulfonic acid compound is added preferably in the range of0.005 to 20 μmol and the nitro compound is added preferably in the rangeof 0.005 to 25 μmol, and the former is added more preferably in therange of 0.02 to 4 μmol and the latter is added more preferably in therange of 0.01 to 5 μmol with respect to 1 μl of the sample.

The conditions of the protease treatment are not particularly limitedbut preferably are set according to the optimal conditions of an enzymeto be used, for example. When the protease is metalloproteinase, thetemperature ranges from 10° C. to 37° C. and the treating period rangesfrom 30 seconds to 60 minutes. It is preferable that the temperatureranges from 20° C. to 37° C. and the treating period ranges from 30seconds to 10 minutes, and it is more preferable that the former rangesfrom 25° C. to 37° C. and the latter ranges from 30 seconds to 5minutes.

Next, as described above, the method for measuring a glycated proteinaccording to the present invention, in which an amount of the glycatedprotein is determined by treating a sample containing the glycatedprotein with a protease so as to degrade the glycated protein, allowinga glycated portion of a glycated protein degradation product obtained bythe degradation and a fructosyl amino acid oxidase to react with eachother, and measuring this redox reaction, includes carrying out theprotease treatment in the presence of a sulfonic acid compound.

In the measuring method of the present invention, the amount of thesulfonic acid compound to be added is not particularly limited but canbe determined suitably according to, for example, the kind and addedamount of the protease, the kind of a sample, the amount of glycatedproteins contained in the sample or the like.

More specifically, the sulfonic acid compound is added preferably in therange of 0.01 to 1000 μmol, more preferably in the range of 0.03 to 200μmol and particularly preferably in the range of 0.05 to 40 μmol withrespect to 1 μL of the sample.

In the case of adding both the sulfonic acid compound and the nitrocompound, the sulfonic acid compound is added preferably in the range of0.005 to 20 μmol and the nitro compound is added preferably in the rangeof 0.005 to 25 μmol, and the former is added more preferably in therange of 0.02 to 4 μmol and the latter is added more preferably in therange of 0.01 to 5 μmol with respect to 1 μl of the sample.

In the measuring method of the present invention, there is no particularlimitation on the samples. Other than blood samples such as whole blood,plasma, serum and blood cells, the samples can be, for example,biological samples such as urine, spinal fluid and saliva, drinks suchas juices, or foods such as soy sauce and Worcestershire sauce. Amongthe above, blood samples such as whole blood and blood cells arepreferable.

The analyte in the present invention, namely, the glycated protein to bedegraded with the protease is, for example, glycated hemoglobin,glycated albumin or the like, and among them, preferably is glycatedhemoglobin. Since hemoglobin in blood has a concentration of as high asabout 60 to 200 g/L and thus is difficult to degrade, the proteasetreatment has taken several hours to several days. The measuring methodof the present invention makes it possible to carry out the proteasetreatment within, for example, 20 seconds to 2 hours, allowing quickmeasurement of the glycated hemoglobin.

Additionally, in the measuring method according to the presentinvention, it is preferable to carry out a protease treatment in thepresence of not only the sulfonic acid compound but also the nitrocompound because the degradation of the glycated protein can beaccelerated further.

In the measuring method according to the present invention, it ispreferable that the redox reaction is measured by determining an amountof hydrogen peroxide generated by the reaction of the glycated portionof the glycated protein and the FAOD. It is also preferable that thisamount of the hydrogen peroxide is determined by using an oxidase suchas POD to reduce the generated hydrogen peroxide and oxidize a substratethat develops color by oxidation (hereinafter, referred to as a“color-developing substrate”) and measuring a degree of the color thatthe substrate has developed.

The color-developing substrate is not particularly limited but can be,for example, color-developing substrates as listed below. Thesecolor-developing substrates usually have an absorbance at 400 nm orlonger. On the other hand, the sulfonic acid compound and the nitrocompound described above generally do not have an absorbance at 400 nmor longer, so that there is no need to worry about the error caused inthe measurement even when used with these color-developing substrates.

More specifically, the color-developing substrate can be, for example,N-(carboxymethylaminocarbonyl) -4,4′-bis(dimethylamino)diphenylaminesodium salt (hereinafter, referred to as “DA-64”), a combination ofTrinder's reagent and 4-aminoantipyrine,N,N,N′,N′,N″,N″,-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethanehexasodium salt (hereinafter, referred to as “TPM-PS”), N,N,N′,N′,N″,N″-hexa(2-hydroxy-3-sulfopropyl)-4,4′,4″-triaminotriphenylmeth anehexasodium salt (hereinafter, referred to as “TPM-OS”),10-(carboxymethylaminocarbonyl)3,7-bis(dimethylamino) phenothiazinesodium salt (hereinafter, referred to as “DA-67”),10-(methylaminocarbonyl)3,7-bis(dimethylamino) phenothiazine(hereinafter, referred to as “MCDP”),10-(carboxyaminomethyl-4-benzaminocarbonyl)3,7-bis(dimethylamino)phenothiazine sodium salt (hereinafter, referred to as “MMX”) or thelike. Among the above, triaminotriphenylmethane-based color-developingsubstrates are preferable, for example.

The Trinder's reagent can be, for example, phenols, phenol derivatives,aniline derivatives, naphthols, naphthol derivatives, naphthylamine ornaphthylamine derivatives. The compound to be combined with theTrinder's reagent may be not only 4-aminoantipyrine noted above butalso, for example, aminoantipyrine derivatives, vanillin diaminesulfonic acid, methyl benzothiazolinone hydrazone (MBTH), sulfonatedmethyl benzothiazolinone hydrazone (SMBTH) or the like.

In the present invention, it is preferable that the FAOD is a FAODcatalyzing a reaction represented by Formula (1) below.R¹—CO—CH₂—NH—R²+H₂O+O₂→R¹—CO—CHO+NH₂—R²+H₂O₂  (1)

In Formula (1), R¹ represents a hydroxyl group or a residue derived fromthe sugar before glycation (i.e., sugar residue). The sugar residue (R¹)is an aldose residue when the sugar before glycation is aldose, and is aketose residue when the sugar before glycation is ketose. For example,when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by—[CH(OH)]_(n)—CH₂OHwhere n is an integer of 0 to 6.

In Formula (1), R² is not particularly limited. However, when thesubstrate is a glycated amino acid, a glycated peptide or a glycatedprotein, for example, there is a difference between the case where anα-amino group is glycated and the case where an amino group other thanthe α-amino group is glycated.

In Formula (1), when an a-amino group is glycated, R² is an amino acidresidue or a peptide residue represented by Formula (2) below.—CHR³—CO—R⁴   (2)

In Formula (2), R³ denotes an amino-acid side chain group. R⁴ denotes ahydroxyl group, an amino acid residue or a peptide residue, and can berepresented, for example, by Formula (3) below. In Formula (3), n is aninteger of 0 or larger, and R³ denotes an amino-acid side chain group asin the above.—(NH—CHR³—CO)_(n)—OH   (3)

In Formula (1) above, when an amino group other than the cc-amino groupis glycated (i.e., an amino-acid side chain group is glycated), R² canbe represented by Formula (4) below.—R⁵—CH(NH—R⁶)—CO—R⁷   (4)

In Formula (4) above, R⁵ denotes a portion other than the glycated aminogroup in the amino-acid side chain group. For example, when the glycatedamino acid is lysine, R⁵ is as follows.—CH₂—CH₂—CH₂—CH₂—

For another example, when the glycated amino acid is arginine, R⁵ is asfollows.—CH₂—CH₂—CH₂—NH—CH(NH₂)—

In Formula (4) above, R⁶ denotes hydrogen, an amino acid residue or apeptide residue, and can be represented, for example, by Formula (5)below. In Formula (5), n denotes an integer of 0 or lager, and R³denotes an amino-acid side chain group as in the above.—(CO—CHR³—NH)_(n)—H   (5)

In Formula (4) above, R⁷ denotes a hydroxyl group, an amino acid residueor a peptide residue, and can be represented, for example, by Formula(6) below. In Formula (6), n is an integer of 0 or lager, and R³ denotesan amino-acid side chain group as in the above.—(NH—CHR³—CO)_(n)—OH   (6)

In the following, the method of measuring a glycated protein accordingto the present invention will be described by way of specific examples.It should be noted that the present invention is by no means limited byan embodiment below.

First Embodiment

The method of measuring a glycated protein according to the presentinvention will be described with reference to examples in which ananalyte is a glycated protein in blood cells.

First, whole blood itself is hemolyzed, or a blood cell fraction isseparated from whole blood in the usual way such as centrifugation andthen hemolyzed, so as to prepare a hemolyzed sample. The method ofcausing the hemolysis is not particularly limited and can be, forexample, a method using a surfactant, a method using ultrasonic waves, amethod utilizing a difference in osmotic pressure or the like. Amongthese, the method using a surfactant is preferable.

The surfactant for hemolysis is not particularly limited, and examplesthereof include nonionic surfactants such aspolyoxyethylene-p-t-octylphenyl ether (e.g. Triton series surfactants),polyoxyethylene sorbitan alkyl ester (e.g. Tween series surfactants),polyoxyethylene alkyl ether (e.g. Brij series surfactants) and the like.Specific examples thereof are trade name Triton X-100, trade nameTween-20, trade name Brij 35, etc. The conditions of the treatment withthe surfactant usually are as follows: when the concentration of bloodcells in the solution to be treated is 1 to 10 vol %, the surfactant isadded so that its concentration in the solution falls in the range from0.1 to 1 wt %, and stirred at room temperature for about 5 seconds to 1minute.

In the case of utilizing the difference in osmotic pressure, thehemolysis can be carried out by, for example, adding purified water in avolume that is 2 to 100 times as much as the whole blood.

Next, the hemolyzed sample is treated with the protease in the presenceof the sulfonic acid compound so as to degrade the glycated protein,thus preparing a glycated protein degradation product. The reason whythe glycated protein is treated with the protease as described abovefollows. Considering the fact that the FAOD to be used in the subsequentprocess is unlikely to act on proteins and long polypeptide chains asdescribed earlier, these proteins and long polypeptide chains aredegraded so that the FAOD can act on the glycated portions more easily.Since the protease treatment in the presence of the sulfonic acidcompound can degrade the glycated protein within a short time and with ahigh degradation efficiency as described above, even a short period ofthe protease treatment allows the FAOD to act on the glycated portionsufficiently. To further accelerate the degradation, the treatment maybe carried out in the presence of the sulfonic acid compound and thenitro compound as described later.

The protease treatment usually is carried out in a buffer. The kind ofthe buffer is not particularly limited but can be, for example, atris-hydrochloric acid buffer, an EPPS buffer, a PIPES buffer, aphosphate buffer, an ADA buffer, a citrate buffer, an acetate buffer, aglycinamide buffer, a CHES buffer or the like. The pH thereof preferablyranges from 5 to 12, more preferably from 6 to 10, and particularlypreferably from 7 to 9.

As the protease, for example, protease K, subtilisin, trypsin,aminopeptidase and the like can be used as described above. The ratio ofthe protease to be added is as follows: for example, when theconcentration of blood cells in the solution to be treated is 0.1 to 10vol %, the protease is added so that its ratio in the solution rangespreferably from 0.1 to 100 g/L, more preferably from 0.3 to 50 g/L andparticularly preferably from 0.5 to 20 g/L.

In the case where the glycated protein to be degraded is glycatedhemoglobin, it is preferable to use as the protease a protease degradingthe glycated hemoglobin selectively, e.g., bromelain, papain, trypsinderived from porcine pancreas, metalloproteinase, and protease derivedfrom Bacillus subtilis, as described earlier. The ratio of the proteaseto be added is as follows: for example, when the concentration of bloodcells in the solution to be treated is 0.1 to 10 vol %, the protease isadded so that its ratio in the solution ranges preferably from 0.1 to 50g/L, more preferably from 0.3 to 30 g/L and particularly preferably from1 to 20 g/L. More specifically, when the protease is metalloproteinase,it preferably is added to the solution with a blood cell concentrationof 0.3 to 5 vol % so that its ratio in the solution ranges from 0.1 to30 g/L, more preferably from 0.3 to 20 g/L and particularly preferablyfrom 1 to 10 g/L.

The ratio of the sulfonic acid compound to be added is as follows: forexample, when the concentration of blood cells in the solution to betreated with the protease is 1 vol %, the sulfonic acid compound isadded so that its concentration in the solution ranges preferably from0.0001 to 100 mmol/L, more preferably from 0.0003 to 60 mmol/L andparticularly preferably from 0.001 to 30 mmol/L. More specifically, whenthe sulfonic acid compound is SLS and the blood cell concentration inthe solution to be treated with the protease is 1 vol %, the sulfonicacid compound preferably is added to the solution so that itsconcentration in the solution ranges from 0.1 to 100 mmol/L, morepreferably from 0.2 to 60 mmol/L and particularly preferably from 0.5 to30 mmol/L.

Although the sulfonic acid compound may be used as it is, it preferablyis dissolved in a solvent and used as a sulfonic acid compound solutionin view of simplicity of operation and treatment efficiency. Theconcentration of this solution can be determined suitably depending onthe kind of the sulfonic acid compound or the like and ranges, forexample, from 1 to 1000 mmol/L. As the solvent, it is possible to usedistilled water, a physiological salt solution, buffers and the like,and the buffer can be any of those listed above, for example.Incidentally, the above-mentioned sulfonic acid compound may be one kindor a combination of two or more.

The conditions of the protease treatment in the presence of the sulfonicacid compound are not particularly limited but, for example, are asfollows: the temperature ranges from 10° C. to 37° C., and the treatingperiod ranges from 30 seconds to 60 minutes. The temperature preferablyranges from 20° C. to 37° C., and the treating period preferably rangesfrom 30 seconds to 10 minutes. The former more preferably ranges from25° C. to 37° C., and the latter more preferably ranges from 30 secondsto 5 minutes.

The protease treatment of a sample in the presence of the sulfonic acidcompound in this manner can further accelerate the degradation of theglycated protein in the sample, so that the degradation product of theglycated protein can be obtained within a short time and with a highdegradation efficiency.

Also, by carrying out this protease treatment in the presence of notonly the sulfonic acid compound but also the nitro compound, thedegradation with the protease can be accelerated further.

The ratio of the nitro compound to be added is not particularly limitedbut can be determined suitably depending on, for example, the amount ofthe sulfonic acid compound to be added, the amount of protease, etc.When the blood cell concentration in the solution to be treated with theprotease is 1 vol %, the nitro compound is added so that, for example,its concentration in the solution ranges preferably from 0.01 to 25mmol/L and more preferably from 0.05 to 10 mmol/L.

Next, the degradation product obtained by the protease treatment istreated with the FAOD. This FAOD treatment catalyzes the reaction shownby Formula (1) above.

It is preferable that the FAOD treatment is carried out in a buffer asin the above protease treatment. The conditions of the FAOD treatmentare determined suitably depending on the kind of the FAOD used, the kindand concentration of the glycated protein as the analyte, etc.

More specifically, the conditions are as follows: the concentration ofthe FAOD in the reaction solution ranges from 50 to 50,000 U/L, theconcentration of blood cells in the reaction solution ranges from 0.01to 1 vol %, a reaction temperature ranges from 15° C. to 37° C., areaction period ranges from 1 to 60 minutes, and a pH ranges from 6 to9. Moreover, the kind of the buffer is not particularly limited, and forexample, the buffers similar to those in the protease treatment can beused.

Subsequently, POD and the color-developing substrate are added to thehydrogen peroxide generated in the above FAOD treatment so as to allowthe substrate to develop color, and the degree of the color developed ismeasured. When the POD is allowed to act on the hydrogen peroxide, thehydrogen peroxide is reduced and the color-developing substrate isoxidized so as to develop color. Since there is a correlation betweenthe degree of color that the substrate has developed by oxidation andthe amount of the generated hydrogen peroxide, the amount of thehydrogen peroxide can be determined by measuring the degree of the colordeveloped.

The color-developing substrate can be substrates as described above andparticularly preferably isN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt.

The color development reaction usually is carried out in a buffer. Theconditions of the reaction are determined suitably depending on theconcentration of the generated hydrogen peroxide, etc. The conditionsare usually as follows: the concentration of the POD in the reactionsolution ranges from 10 to 20,000 IU/L, the concentration of thecolor-developing substrate ranges from 0.001 to 1 mmol/l, a reactiontemperature ranges from 20° C. to 37° C., a reaction period ranges from1 to 5 minutes, and a pH ranges from 6 to 9. Moreover, the kind of thebuffer is not particularly limited, and for example, the buffers similarto those in the protease treatment and the FAOD treatment can be used.

In the color development reaction, for example, when thecolor-developing substrate is used, the amount of the hydrogen peroxidecan be determined by measuring the degree of the color developed (i.e.absorbance) in the reaction solution with a spectrophotometer. Then,using this concentration of the hydrogen peroxide and a calibrationcurve or the like, the amount of the glycated protein in the sample canbe determined.

The amount of the hydrogen peroxide can be determined not only by theabove-described enzymatic method using a POD or the like but also by anelectrical method, for example.

With such a measuring method according to the present invention, it ispossible to perform measurements quickly as described above. Also, theconventional method may suffer from a lowered accuracy of measurementwhen the protease treatment is shortened, whereas the measuring methodof the present invention achieves a measurement with an excellentaccuracy even within a short period.

EXAMPLES

Hereinafter, examples will be described together with comparativeexamples.

Example 1, Comparative example 1

In Example 1, glycated hemoglobin was treated with a protease in thepresence of a sulfonic acid compound, thus measuring the degree ofglycation of its degradation product using a color-developing substrateTPM-PS. The sample and reagents used here and the method will bedescribed below. The sulfonic acid compound in a first reagent describedbelow was SLS (manufactured by Nacalai Tesque, Inc.).

(Preparation of Sample to be Measured)

Lyophilized hemoglobin was dissolved in purified water, thus preparing ahemoglobin solution with a hemoglobin concentration of 50 g/L and aHbAlc concentration of 6.5% (HbAlc: Low) and a hemoglobin solution witha hemoglobin concentration of 50 g/L and a HbAlc concentration of 11.5%(HbAlc: High). Then, 60 μL of each of these hemoglobin solutions wasmixed with 240 μL of purified water so as to prepare a sample with aHbAlc concentration of 6.5% (hereinafter, referred to as a “sample Low”)and a sample with a HbAlc concentration of 11.5% (hereinafter, referredto as a “sample High”).

(First Reagent)

Sulfonic acid compound 6.4 mM Surfactant (polyoxyethylene(9)dodecylether) 1.85 g/L CHES-CHES · Na buffer (pH 9.4) 40 mM MOPS-MOPS · Nabuffer (pH 9.4) 15 mM(Second Reagent)

Metalloproteinase (manufactured by ARKRAY, INC.) 2.0 MU/L CaCl₂ 2.5 mMNaCl 50 mM MOPS-MOPS · Na buffer (pH 6.5) 1.0 mM(Third Reagent)

FAOD (manufactured by ARKRAY, INC.) 18 KU/L POD (manufactured byKikkoman Corporation) 67 KU/L TPM-PS (manufactured by DOJINDOLABORATORIES) 0.25 mM Tris-HCl buffer (pH 7.0) 300 mM(Method)

Measurement was carried out for each of the sample Low and the sampleHigh. First, after 8.26 μL of the first reagent was added to 0.14 μL ofthe sample to be measured, 75.6 μL of the second reagent further wasmixed therein and allowed to stand at 37° C. for 5 minutes. Then, 18.9μL of the third reagent was blended into this mixture and incubated at37° C. so as to allow a color development reaction. The absorbance ofthe reaction solution 2.5 minutes after adding the third reagent wasmeasured with trade name JCA-BM8 (manufactured by JEOL. Ltd.). Themeasurement wavelengths were set to 571 nm for the main wavelength and805 nm for the sub-wavelength. On the other hand, in the ComparativeExample, the absorbance was measured similarly to Example 1 describedabove except that the sulfonic acid compound in the first reagent wasnot added. When the first reagent and the second reagent were mixed intothe sample, the amount of the added sulfonic acid compound was 0.378μmol with respect to 1 μL of the sample.

TABLE 1 Sulfonic Absorbance acid Nitro Sample Sample compound compoundHigh Low High − Low (mM) (mM) (mAbs) (mAbs) (mAbs) Example 1 SLS (6.4) —51.0 34.0 17.0 Comparative — — 16.7 16.0 0.7 example 1

As shown in Table 1 above, in Example 1, the absorbance of the sampleHigh and the absorbance of the sample Low respectively were higher thanthose of the Comparative Example. This is because, by the proteasetreatment in the presence of the sulfonic acid compound, the degradationof the glycated hemoglobin was accelerated, so that the FAOD could acton glycated portions more easily. In other words, according to Example1, the acceleration of the degradation allowed the FAOD to act easily onmore glycated portions than the Comparative Example, thus improving theaccuracy of measurement.

Further, in Example 1, the difference between the absorbance of thesample High and that of the sample Low was greater than that in theComparative Example. The sample High had a hemoglobin concentrationequal to the sample Low but had a higher HbAlc concentration than thesample Low. In other words, because of a greater glycated amount ofhemoglobin, the sample High theoretically had an absorbance greater thanthe sample Low in proportion to the HbAlc concentration. However, in theComparative Example, since the protease treatment was carried out in theabsence of the sulfonic acid compound, the glycated hemoglobin wasdifficult to degrade. Therefore, even though the sample High and thesample Low had different HbAlc concentrations, their difference inabsorbance was as small as 0.7 mAbs. On the other hand, in the Examplewhere the protease treatment was carried out in the presence of thesulfonic acid compound, the difference between the absorbance of thesample High and that of the sample Low increased to about twenty timesto thirty times that in the Comparative Example. This also shows that,for the same reason as above, the method according to Example 1 improvedthe sensitivity and accuracy of measurement.

Example 2

In Example 2, glycated hemoglobin was treated with a protease in thepresence of a sulfonic acid compound, thus measuring the degree ofglycation of its degradation product using a color-developing substrateDA-64. The sample and reagents used here and the method will bedescribed below.

(First Reagent)

Sulfonic acid compound (SLS: manufactured by Nacalai 6.4 mM Tesque,Inc.) Surfactant (polyoxyethylene(9)dodecyl ether) 1.85 g/L CHES-CHES ·Na buffer (pH 9.4) 40 mM MOPS-MOPS · Na buffer (pH 9.4) 15 mM(Second Reagent)

Nitro compound 0.9 mM or 1.8 mM Metalloproteinase (manufactured by 2.0MU/L ARKRAY, INC.) CaCl₂ 2.5 mM NaCl 50 mM MOPS-MOPS · Na buffer (pH6.5) 1.0 mM

As the nitro compound, 2,4-DNA (manufactured by Wako Pure ChemicalIndustries, Ltd.), p-NA (manufactured by Wako Pure Chemical Industries,Ltd.), p-NP (manufactured by Wako Pure Chemical Industries, Ltd.), NaNO2(manufactured by Nacalai Tesque, Inc.) and 2,4-DNH (manufactured by WakoPure Chemical Industries, Ltd.) were used individually. In the case ofadding two kinds of the nitro compound, the concentration was 1.8 mM intotal (0.9 mM each).

(Third Reagent)

FAOD (manufactured by ARKRAY, INC.) 18 KU/L POD (manufactured byKikkoman Corporation) 67 KU/L DA-64 (manufactured by Wako Pure Chemical70 μM Industries, Ltd.) Tris-HCl buffer (pH 7.0) 300 mM(Method)

Measurement was carried out for each of the sample Low and the sampleHigh that were prepared in Example 1 described above. After 8.26 μL ofthe first reagent was added to 0.14 μL of the sample to be measured,75.6 μL of the second reagent further was mixed therein and allowed tostand at 37° C. for 5 minutes. Then, 18.9 μL of the third reagent wasblended into this mixture and incubated at 37° C. so as to allow a colordevelopment reaction. The absorbance after 5 minutes was measured withtrade name JCA-BM8 (manufactured by JEOL. Ltd.). The measurementwavelengths were set to 751 nm for the main wavelength and 805 nm forthe sub-wavelength. On the other hand, in Comparative example 2, theabsorbance was measured similarly to Example 1 described above exceptthat the sulfonic acid compound in the first reagent and the nitrocompound in the second reagent were not added. The results are shown inTable 2 below. In Table 2, “High-Low (mAbs)” is a value obtained bysubtracting the absorbance of the sample Low from that of the sampleHigh.

TABLE 2 Absorbance Sulfonic acid Nitro Sample Sample High − compoundcompound High Low Low Example (mM) (mM) (mAbs) (mAbs) (mAbs) 2-1 SLS(6.4) 2,4-DNA (0.9) 4.2 1.6 2.6 2-2 SLS (6.4) p-NA (0.9) 2.6 1.2 1.4 2-3SLS (6.4) p-NP (0.9) 3.2 1.6 1.6 2-4 SLS (6.4) NaNO₂ (0.9) 2.3 1.3 1.02-5 SLS (6.4) 2,4-DNA (0.9) 3.5 1.6 1.9 p-NA (0.9) 2-6 SLS (6.4) 2,4-DNA(0.9) 2.8 1.1 1.7 p-NA (0.9) 2-7 SLS (6.4) p-NP (0.9) 3.3 1.3 2.0 p-NA(0.9) 2-8 SLS (6.4) 2,4-DNA (0.9) 4.0 1.9 2.1 NaN₃ (0.9) Comp. — — 1.10.9 0.2 example 2

As shown in Table 2 above, in Example 2, the absorbance of the sampleHigh and the absorbance of the sample Low respectively were higher thanthose of the Comparative Example, similarly to Example 1. Also, thedifference between the absorbance of the sample High and that of thesample Low was greater than that in Comparative Example 2. These resultsshow that, similarly to Example 1 above, by the protease treatment inthe presence of the sulfonic acid compound and the nitro compound, thedegradation of the glycated hemoglobin was accelerated, thus improvingthe sensitivity and accuracy of measurement.

INDUSTRIAL APPLICABILITY

As described above, in accordance with the measuring method according tothe present invention, by a protease treatment in the presence of thesulfonic acid compound, proteins and the like can be degraded within ashort time, thus allowing a quick measurement of glycated proteins andthe like. This makes it possible to achieve a measurement with a stillhigher accuracy, and accordingly, the measuring method according to thepresent invention is useful for various tests in clinical medicine asdescribed above.

1. A method of measuring the amount of a glycated protein in a sample,the method comprising: treating a sample containing the glycated proteinwith a protease in the presence of a sulfonic acid compound and a nitrocompound to accelerate the degradation of the glycated protein, adding afructosyl amino acid oxidase to react in a redox reaction with aglycated portion of a glycated protein degradation product obtained bythe protease treatment, and measuring the redox reaction, wherein thesulfonic acid compound is at least one selected from the groupconsisting of 4-aminoazobenzene-4′-sulfonic acid sodium salt,4-amino-4′-nitrostilbene-2,2′-disulfonic acid disodium salt,4,4′-diazidostilbene-2,2′-disulfonic acid disodium salt,N-cyclohexyl-2-aminoethane sulfonic acid, N-cyclohexyl-3-aminopropanesulfonic acid, N-cyclohexyl-2-hydroxy-3-aminopropane sulfonic acid,piperazine-1,4-bis(2-ethane sulfonic acid) and bathophenanthrolinesulfonic acid, wherein the nitro compound is at least one selected fromthe group consisting of 2,4-dinitrophenol, 2,5-dinitrophenyl,2,6-dinitrophenyl, 4,6-dinitro-2-methyl phenol, 2-amino-4-nitrophenol,2-amino-5-nitrophenol, 2-amino-4-nitrophenol, p-nitrophenol,2,4-dinitroaniline, p-nitroaniline,4-amino-4′-nitrostilbene-2,2′-disulfonic acid disodium salt andnitrobenzene, wherein the redox reaction is measured by determining anamount of hydrogen peroxide generated by the reaction of the glycatedportion of the glycated protein degradation product and the frucytosylamino acid oxidase, and wherein the amount of the hydrogen peroxide isdetermined by using an oxidase to reduce the generated hydrogen peroxideand oxidize a substrate that develops color by oxidation and measuring adegree of the color that the substrate has developed.
 2. The methodaccording to claim 1, wherein the protease is a metalloproteinase. 3.The method according to claim 1, wherein the degree of the color ismeasured by measuring an absorbance at a wavelength for detecting thesubstrate.